Breast cancer is the most common cancer in women and the second leading cause of cancer death in women. The peptide hormone prolactin (PRL) promotes normal breast tissue growth and maturation, but PRL also contributes to breast cancer development. The long-term goal of this application is to determine molecular mediators of PRL signaling which can serve as targets for novel therapeutics. PRL signals by binding to the transmembrane PRL receptor (PRLr). The PRLr signals from the cell surface to the nucleus in two ways, 1) by activating canonical signaling pathways and 2) by translocating to the nucleus. In the nucleus, the PRLr promotes gene expression, but the mechanism by which it does so has not been fully elucidated. We have shown that nuclear PRLr binds to the chromatin-modifying protein high-mobility group N2 (HMGN2), which is also important in promoting the expression of PRL-responsive genes. HMGN2 can mediate chromatin decompaction, which may promote gene expression by allowing the transcriptional machinery to access the promoter DNA. Consistent with mediating chromatin decompaction, HMGN2 has been shown to promote histone acetylation and displacement of the chromatin-condensing linker histone H1. The enclosed aims will determine the mechanism by which HMGN2 promotes the transcription of PRL- responsive genes. The extent of HMGN2-mediated alterations at the promoters of PRL-responsive genes, including histone acetylation and H1 displacement, will be investigated by chromatin immunoprecipitation (ChIP). The mechanism by which HMGN2 promotes such changes, such as by recruiting histone acetyltransferases, will also be investigated. In addition, the enclosed aims will determine how HMGN2 activity is regulated. HMGN2 appears to be regulated by post-translational acetylation. HMGN2 mutants mimicking or preventing such modification will be analyzed for their ability to mediate chromatin decompaction, which will be investigated using ChIP to identify relevant histone modifications at gene promoters. The ability of acetylated HMGN2 to bind to the PRLr will also be assessed by co-immunoprecipitation. The rationale for the proposed research is that by determining how HMGN2 promotes transcription and how that process is regulated, various proteins and processes will be implicated as targets for novel breast cancer therapeutics to attenuate PRLr signaling. PUBLIC HEALTH RELEVANCE: The proposed research aims to establish the role and regulation of HMGN2 in promoting gene expression, which will provide a mechanistic link between nuclear translocation of the PRLr and breast cancer pathogenesis. Characterization of this novel PRLr signaling pathway will have a positive impact on public health by improving our understanding of breast cancer pathogenesis and by identifying targets for the development of novel breast cancer therapies, which are desperately needed for this common and deadly disease.